APPLIED NUTRITIONAL INVESTIGATION INTRODUCTION MATERIALS AND METHODS

APPLIED NUTRITIONAL INVESTIGATION Cardiovascular Effects of Milk Enriched With ␻-3 Polyunsaturated Fatty Acids, Oleic Acid, Folic Acid, and Vitamins ...
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APPLIED NUTRITIONAL INVESTIGATION

Cardiovascular Effects of Milk Enriched With ␻-3 Polyunsaturated Fatty Acids, Oleic Acid, Folic Acid, and Vitamins E and B6 in Volunteers With Mild Hyperlipidemia Juan J. Carrero, MSc, Luis Baro´, PhD, Juristo Fonolla´, PhD, Marı´a Gonza´lez-Santiago, MSc, Antonio Martı´nez-Fe´rez, PhD, Rafael Castillo, MD, Jesu´s Jime´nez, PhD, Julio J. Boza, PhD, and Eduardo Lo´pez-Huertas, PhD From Puleva Biotech SA, Departamento de Ingenierı´a Quı´mica, Universidad de Granada, and Hospital Universitario “San Cecilio,” Granada, Spain OBJECTIVE: Results from epidemiologic studies and clinical trials have indicated that consumption of ␻-3 fatty acids, oleic acid, and folic acid have beneficial effects on health, including decreased risk of cardiovascular disease. We evaluated the combined effects of these nutrients through the consumption of milk enriched with ␻-3 polyunsaturated fatty acids, oleic acid, vitamins E and B6, and folic acid on risk factors for cardiovascular disease in volunteers with mild hyperlipidemia. METHODS: Thirty subjects ages 45 to 65 y (51.3 ⫾ 5.3 y) were given 500 mL/d of semi-skimmed milk for 4 wk and then 500 mL/d of the enriched milk for 8 wk. Plasma and low-density lipoproteins were obtained at the beginning of the study and at 4, 8, and 12 wk. RESULTS: Consumption of enriched milk for 8 wk increased plasma concentrations of docosahexaenoic acid and eicosapentaenoic acid and significantly (P ⬍ 0.05) decreased plasma concentrations of triacylglycerol (24%), total cholesterol (9%), and low-density lipoprotein cholesterol (13%). Plasma and low-density lipoprotein oxidation and vitamin E concentration remained unchanged throughout the study. Significant decreases in plasma concentrations of vascular cell adhesion molecule-1 (9%) and homocysteine (17%) were found, accompanied by a 98% increase in plasma concentration of folic acid. CONCLUSIONS: Dairy supplementation strategies with ␻-3 polyunsaturated fatty acids, oleic acid, and vitamins may be useful for decreasing risk factors for cardiovascular disease. Nutrition 2004;20: 521–527. ©Elsevier Inc. 2004 KEY WORDS: enriched milk, ␻-3 fatty acids, folic acid, homocysteine, cardiovascular disease

INTRODUCTION There is a wealth of evidence from epidemiologic and clinical studies suggesting that modifications of dietary fat composition affect the risk of cardiovascular disease (CVD).1 Consumption of ␻-3 polyunsaturated fatty acids (␻-3 PUFAs), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has several beneficial properties that prevent CVD, including antiinflammatory, antiarrhythmic, and antihypertensive effects, and are especially valued for their capacity to decrease blood lipids, inhibit the synthesis of cytokines and mitogens, modulate endothelial function, stimulate endothelial-derived nitric oxide, and inhibit atherosclerosis and thrombosis.2–5 Olive oil also is considered a healthy source of fat, and international nutritional guidelines recommend its consumption due to the cardiovascular beneficial effects reported. Supplementation with certain nutrients such as folic acid and vitamins B6 and B12 also has come to be regarded as potentially protective against CVD. For instance, plasma homocysteine concentration, a novel risk factor for CVD, is decreased when the intake of these vitamins is increased.6 Health authorities have recommended increased consumption of PUFAs,7 in which fish oil is especially rich. The most recent

Correspondence to: Eduardo Lo´pez-Huertas, PhD, Puleva Biotech SA, 66, Camino de Purchil, Granada 18004, Spain. E-mail: elopezhuertas@ pulevabiotech.es Nutrition 20:521–527, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.

report by the World Health Organization8 recommends regular fish consumption to provide approximately 200 to 500 mg/wk of EPA and DHA, replacement of saturated fat by monounsaturated fat, and increased consumption of fruits and vegetables to achieve proper antioxidant and vitamin status. However, modern Western societies tend to include very little fish, vegetables, and fruits in their diets, so ways to increase consumption of PUFAs and folic acid have to be explored and assessed at a community or clinical level. An oil blend containing ␻-3 PUFAs, olive oil, vitamins B6 and E, and folic acid was produced and included in skimmed milk to create a dairy product with the palatability of semi-skimmed milk but with a healthier fatty acid and vitamin profile. Milk, an everyday drink, is a very efficient vehicle for absorption of fat and lipid-soluble compounds because of its dispersion in micelles. In this 8-wk study, we tested the hypothesis that the substitution of regular milk (approximately 70% saturated fat) with this dairy product would have the potential to decrease cardiovascular risk factors in free-living, mildly hyperlipidemic subjects.

MATERIALS AND METHODS To ensure analytical consistency, samples at the beginning of the study (T⫺4) and at 8 wk (T8) from the same volunteers were processed at the same time and analyzed in one batch when techniques involving high-performance liquid chromatography, gas-liquid chromatography, or spectrophotometry were used. For 0899-9007/04/$30.00 doi:10.1016/j.nut.2004.03.017

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Nutrition Volume 20, Number 6, 2004 TABLE I.

CALCULATED DIETARY INTAKE OF NUTRIENTS AT BASELINE AND AT WEEK 11 IN THE EXPERIMENTAL PERIOD EXCLUDING THE MILKS TESTED* Week 1

Energy (kcal) Proteins (g) Carbohydrates (g) Total fat (g) SFAs (g) MUFAs (g) PUFAs (g) Fiber (g) Calcium (mg) Iron (mg) Sodium (mg) Vitamin A (␮g) Thiamin (mg) Riboflavin (mg) Vitamin B6 (mg) Vitamin B12 (␮g) Vitamin C (mg) Vitamin D (␮g) Vitamin E (mg) Niacin (mg) Folate (␮g)

Week 11

Men (n ⫽ 15)

Women (n ⫽ 15)

Men (n ⫽ 15)

Women (n ⫽ 15)

2398 ⫾ 46 96.1 ⫾ 3.2 257.4 ⫾ 6.2 98.1 ⫾ 4.5 32.5 ⫾ 1.0 42.6 ⫾ 0.8 14.4 ⫾ 0.4 17.6 ⫾ 0.5 874.5 ⫾ 10.2 13.2 ⫾ 0.2 1942.5 ⫾ 16.6 865 ⫾ 97 1.21 ⫾ 0.06 2.0 ⫾ 0.1 2.20 ⫾ 0.09 11.1 ⫾ 0.72 125.4 ⫾ 14.3 4.8 ⫾ 0.68 9.8 ⫾ 0.35 29.8 ⫾ 0.54 199.4 ⫾ 6.4

1880 ⫾ 32 78.1 ⫾ 1.8 205.4 ⫾ 5.0 77.7 ⫾ 2.0 24.4 ⫾ 0.9 31.5 ⫾ 0.8 10.4 ⫾ 0.2 16.0 ⫾ 0.4 800.6 ⫾ 9.5 10.4 ⫾ 0.1 1393.2 ⫾ 20.8 857 ⫾ 74 1.10 ⫾ 0.09 1.8 ⫾ 0.3 1.75 ⫾ 0.10 8.4 ⫾ 0.65 137.4 ⫾ 20.8 3.4 ⫾ 0.34 7.2 ⫾ 0.47 22.7 ⫾ 1.5 196.7 ⫾ 7.9

2412 ⫾ 37 95.2 ⫾ 1.5 265 ⫾ 5.8 97.2 ⫾ 3.6 31.1 ⫾ 0.9 41.6 ⫾ 0.9 14.9 ⫾ 0.3 17.6 ⫾ 0.3 880 ⫾ 9.8 13.5 ⫾ 0.4 1950 ⫾ 34.2 882 ⫾ 103 1.14 ⫾ 0.08 1.7 ⫾ 0.2 2.29 ⫾ 0.11 10.8 ⫾ 0.86 145.2 ⫾ 25.2 5.4 ⫾ 0.84 9.6 ⫾ 0.89 31.1 ⫾ 0.86 189.2 ⫾ 8.4

1869 ⫾ 24 77.3 ⫾ 1.8 202.8 ⫾ 3.4 79.3 ⫾ 1.9 24.7 ⫾ 1.0 31.8 ⫾ 0.6 10.7 ⫾ 0.3 15.8 ⫾ 0.2 807 ⫾ 11.3 10.6 ⫾ 0.6 1375 ⫾ 24.8 825 ⫾ 74 1.16 ⫾ 0.10 1.6 ⫾ 0.6 1.84 ⫾ 0.14 9.1 ⫾ 0.72 128.4 ⫾ 14.7 3.1 ⫾ 0.47 8.1 ⫾ 0.51 23.7 ⫾ 64 200.0 ⫾ 5.9

* Average ⫾ standard error of the mean (n ⫽ 30). SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids

enzyme-linked immunosorbent assay determinations, all analyzed samples were processed and run in one batch. Subjects and Study Design Thirty subjects (15 men and 15 women; age range, 45– 65 y, 51.3 ⫾ 5.3 y) were recruited in Granada (Spain) from volunteers who responded to an advertisement about dietary intervention studies. We advertised for subjects ages 45 to 65 y, preferably with high levels of blood triacylglycerols. Subjects were given a physical examination, and their medical histories were taken before they were included in the study. Subjects without chronic or metabolic disease and not taking medications known to influence lipid metabolism from at least 1 mo before the beginning of the study until the end of it were included. Subjects were instructed not to change their physical activities or their usual diets but to avoid eating fish from the beginning until the end of the study. The study was conducted according to the Helsinki Declaration, the protocol was approved by the ethical committee of Puleva Biotech SA, and written informed consent was obtained from all subjects. Dietary intake was assessed at baseline and at week 11 of the study with a 7-d self-administered food-frequency questionnaire. Subjects also were requested to fill in a food diary according to instructions from the principal investigator, where they recorded all food consumption during the study. Compliance with consumption of the study product during the intervention period was ensured and checked by regular telephone calls and weekly collection of the containers consumed. Dietary analysis of the average intake of nutrients during the 12-wk intervention period is presented in Table I, using food composition tables9 as reference values. The milks used in the study were a semi-skimmed milk enriched with vitamins A and D and a commercial dairy product

(Puleva Omega 3, Puleva Food SL, Granada, Spain) containing ␻-3 PUFAs, oleic acid, folic acid, and vitamins A, D, E, and B6 (enriched milk). The dairy product was prepared by adding a mixture of fish and vegetable oils to skimmed milk, resulting in a product containing total fat comparable to that contained in standard semi-skimmed milk (1.9 g/100 mL), but with a different fatty acid profile. Vitamins A, D, E, and B6 and folic acid were added to the final product. The composition and the fatty acid profile of the semi-skimmed milk and the enriched milk are presented in Table II. Subjects drank 500 mL/d of semi-skimmed milk from the beginning of the study (T⫺4) for 4 wk (T0). At T0, subjects replaced the semi-skimmed milk with 500 mL/d of the enriched milk described above, which was consumed for 8 wk. After an overnight fast of 10 h, blood samples (30 mL each) were taken at T⫺4 (beginning of the study), T0 (after consumption of semiskimmed milk for 4 wk), and then at T4 and T8 (after consumption of enriched milk for 4 and 8 wk, respectively). Plasma and Low-Density Lipoprotein Isolation Blood was withdrawn into Vacutainers (S-Monovette, Sarstedt, Germany) containing ethylene-diaminetetra-acetic acid. Plasma was obtained by centrifugation at 3500g for 5 min at 4°C and immediately frozen at ⫺80°C until further analyses. For isolation of low-density lipoprotein (LDL), 10 mL of fresh plasma was transferred to ultracentrifugation tubes and LDL fractions were isolated as described by Chung et al.10 LDL particles typically sedimented at density of 1.006 to 1.063 g/mL. LDL fractions were pooled and dialyzed in the dark for 24 h in 10 mM phosphate buffered saline and 0.15 M NaCl (pH 7.4) and frozen at ⫺80°C under nitrogen atmosphere until needed.

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TABLE II. COMPOSITION OF SEMI-SKIMMED MILK AND ENRICHED MILK, SHOWING RELATIVE FATTY ACID COMPOSITION OF SPECIFIC FATTY ACIDS (AS TOTAL WEIGHT PERCENTAGE IN MILK FAT) AND AMOUNTS OF SFA, MUFA, AND PUFA

Energy (kcal/100 mL) Protein (g/100 mL) Carbohydrates (g/100 mL) Fat (g/100 mL) 18:1 (%) 18:3␻-3 (%) 20:5␻-3 (%) 22:6␻-3 (%) SFA (%) MUFA (%) PUFA (%) Calcium (mg/100 mL) Vitamin A (␮g/100 mL) Vitamin D (␮g/100 mL) Vitamin E (mg/100 mL) Vitamin B6 (mg/100 mL) Vitamin B12 (␮g/100 mL) Folic acid (␮g/100 mL)

Semi-skimmed milk

Enriched milk

46.5 3.1 4.7 1.9 21.5 U U U 70.5 27.2 2.3 120 120 0.75 U U 0.38 U

52 3.5 5.2 1.9 54.4 0.6 1.4 2.1 23.7 56.8 19.5 132 120 0.75 1.50 0.30 0.38 30

MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; U, undetected

Oxidized LDL and Lag-Time Measurements Oxidized LDL in plasma was quantified with a commercial enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden) according to the manufacturer’s instructions. For lag-time measurements, 50 ␮g of protein of dialyzed LDL in 1 mL of phosphate buffered saline was incubated with 10 ␮M CuSO4 for several hours at 30°C. Formation of conjugated dieners was monitored continuously by measuring the increase in absorbance at 234 nm every 10 min. Lag time was determined according to the method of Esterbauer et al.11 Total Antioxidant Capacity and Malondialdehyde Total antioxidant capacity was measured in plasma by using Trolox as the standard. Briefly, 20 ␮L of fresh plasma was 1:1 diluted in phosphate buffered saline and incubated with 1 mL of 2,2⬘-azino-bis(3-ethylbenzo-thiazoline)-b-sulfonic acid (ABTS)⫹ for 20 min. Absorbance was read at 734 nm. ABTS cation was prepared by addition of 88 ␮L of 140 mM potassium persulfate to 5 mL of a 7 mM solution of ABTS in water and incubation for 12 to 14 h. The working solution was obtained by dilution of the former with phosphate buffered saline until the absorbance at 734 nm was 0.7 ⫾ 0.02 as described by Pellegrini et al.12 Plasma malondialdehyde concentrations were measured by separation with high-performance liquid chromatography as described by Fukunaga et al.,13 which is based on the thiobarbituric acid reaction and reverse-phase separation with fluorescence detection. Plasma Lipids, Total Homocysteine, Vitamin E, Plasma Folate, Lipoprotein(a), Vascular Cell Adhesion Molecule-1, and Intercellular Adhesion Molecule-1 Determinations Plasma triacylglycerols, cholesterol, and HDL cholesterol were measured at the hospital central laboratory. All plasma samples

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from subjects were thawed at the same time before analysis. Blood lipids were measured by colorimetry in triplicate in one batch for each determination with commercial reagents obtained from Biosystems (Barcelona, Spain). Plasma fatty acid profile was determined by gas-liquid chromatography as described by Lepage and Roy.14 Total fasting plasma homocysteine (tHcy) concentration was measured by high-performance liquid chromatography with fluorescence detection.15 Plasma vitamin E concentration was determined by high-performance liquid chromatography with ultraviolet detection according to the method described by Thurnham et al.16 Lipoprotein(a) (Lp[a]) levels were quantified by using a commercial kit based on kinetic nephelometry (LPAX Immunochemical Systems, IMMAGE, Beckman Coulter, Hialeah, FL, USA). Plasma folate concentration was measured by immunoassay (SimulTRAC-SNB Radioassay Kit, ICN Pharmaceuticals, Costa Mesa, CA, USA). Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured with commercial enzyme-linked immunosorbent assay kits from Biosource International (Camarillo, California, USA) according to the manufacturer’s instructions. Statistical Analysis All data are expressed as mean ⫾ standard error of the mean. Comparisons across time were assessed by a one-way analysis of variance. When this analysis indicated a significant difference (P ⬍ 0.05), paired Student’s t test followed by Bonferroni’s correction for multiple comparisons were performed. Data were analyzed with SPSS 10.1 for Windows 10.1 (SPSS Chicago, IL, USA).

RESULTS The milks used in the study were well tolerated and compliance was good. No gender differences were found in the parameters measured; therefore, all data are presented as pooled. There was no significant change in body weight change throughout the study (72.74 ⫾ 2.32 kg at T⫺4 versus 72.52 ⫾ 2.37 kg at T8). Dietary Intake of Fatty Acids Total amounts of oleic acid, DHA, and EPA supplemented in 500 mL of the enriched milk were 5.12 g, 0.13 g, and 0.2 g, respectively; the semi-skimmed milk contained only 1.82 g of oleic acid per 500 mL and no detectable levels of DHA and EPA. The enriched milk contained more than eight times the amount of PUFAs and more than twice the amount of monounsaturated fatty acids than the semi-skimmed milk used in the study. Amounts of saturated fatty acids detected in the enriched milk were approximately one-third the amounts in the semi-skimmed milk. Excluding the test milks, the diet consumed by subjects during the study contained negligible levels of DHA and EPA. Dietary contributions calculated for oleic acid and ␣-linolenic acid from foods other than the milks were 23 g/d and 500 mg/d, respectively, on average (Table I). Lipid Profile Average values of major plasma fatty acids detected in subjects are listed in Table III. Average plasma values of lipids before intervention were borderline to high, as defined by the National Cholesterol Education Programme’s Adult Treatment Panel III.17 Interim analysis of blood samples at T⫺4 showed that none of the subjects had lipid values in the range advised for pharmacologic treatment. Consumption of the corresponding semi-skimmed milk during the first 4 wk did not change the plasma fatty acid profile with the exception of a significant decrease in EPA concentration. However, the 8-wk supplementation with the enriched milk not

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Nutrition Volume 20, Number 6, 2004 TABLE III.

VALUES OF MAJOR PLASMA FATTY ACIDS (AS TOTAL PERCENTAGE) FOUND IN SUBJECTS AT DIFFERENT TIME POINTS* T⫺4

T0

T4

T8

Fatty acid

Average

SEM

Average

SEM

Average

SEM

Average

SEM

C16:0 C16:1␻-7 C18:0 C18:1␻-9 C18:1␻-7 C18:2␻-6 C18:3␻-3 C20:3␻-3 C20:4␻-6 C22:5␻-3 EPA DHA

20.05a 1.00 7.32 20.66 1.34 26.41 0.32 1.55 8.25 0.81 0.56a 1.83a

0.39 0.08 0.17 0.84 0.03 0.75 0.02 0.07 0.47 0.09 0.05 0.09

19.93a 1.24 7.39 21.14 1.42 25.94 0.34 1.89 8.43 0.67 0.39b 1.83a

0.24 0.08 0.20 0.71 0.03 0.63 0.03 0.11 0.45 0.07 0.03 0.09

19.36b 1.19 7.27 21.03 1.37 25.56 0.32 1.75 8.54 0.69 0.68c 2.02b

0.30 0.08 0.21 0.77 0.03 0.54 0.02 0.08 0.41 0.07 0.06 0.07

19.22b 1.20 7.24 20.97 1.35 26.21 0.36 1.61 8.42 0.72 0.75c 2.20b

0.21 0.08 0.23 0.68 0.02 0.57 0.02 0.09 0.37 0.07 0.08 0.06

* Average ⫾ SEM (n ⫽ 30). Values with different superscript letters are significantly different (P ⬍ 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; SEM, standard error of the mean; T⫺4, initial values; T0, after 4-wk consumption of semiskimmed milk; T4, after 4-wk consumption of enriched milk; T8, after 8-wk consumption of enriched milk

only restored initial levels of plasma EPA, but a significant (P ⬍ 0.05) increase of 33% was found at T8, as was a sustained 20% increase (P ⬍ 0.05) in DHA plasma concentration. Plasma concentrations of the other fatty acids measured did not change at the time points tested, except for a significant decrease in palmitic acid (C16:0). Fatty acid composition in LDL particles also was measured at different times during the study, but no significant change in fatty acid profile was found in comparison with the fatty acid composition of plasma (not shown). Plasma lipid parameters measured at the different time points of the study are presented in Table IV. The 4-wk consumption of semi-skimmed milk produced a small increase in triacylglycerols, probably as a result of the saturated fat contained in this type of milk (approximately 70%). Consumption of the enriched milk for 8 wk was associated with a significant decrease in total cholesterol of about 9% (P ⬍ 0.05). The effect on LDL cholesterol was more pronounced as the concentration was decreased at T8 by more than 13% (P ⬍ 0.05). Consumption of enriched milk also produced a mild but non-significant linear increase on HDL cholesterol concentration at T4 and T8. With regard to triacylglycerol concentration in plasma, consumption of the enriched milk for 8 wk produced a 24% (P ⬍ 0.05) decrease at the time points compared with the initial levels at T⫺4.

Vitamin E, Plasma, and LDL Oxidation Parameters Malondialdehyde, total antioxidant capacity, and vitamin E were measured in plasma (Table V). No significant differences were found in any of these parameters at the times of the study. To study the effect of ␻-3 PUFA supplementation on LDL oxidation, LDL particles were isolated from subjects, and lag time, or the time required for an oxidant (copper) to induce the propagation phase of oxidation, was measured. We also measured levels of oxidized LDL in plasma by using a monoclonal antibody raised against oxidized LDL. None of these parameters changed at the times tested. Plasma Concentration of ICAM-1, VCAM-1, Lp(a), Folate, and tHcy Plasma concentration of Lp(a) and soluble forms of ICAM-1 and VCAM-1 at the different time points are presented in Table V. We observed a significant 30% (P ⬍ 0.05) decrease in VCAM-1 concentration after the 8-wk consumption of enriched milk (T8) compared with values at T0. ICAM-1 and Lp(a) concentrations measured in plasma remained unchanged throughout the study. Subjects consumed 150 ␮g/d of folic acid from the enriched milk. Increases in plasma folate concentration (P ⬍ 0.05) of 88% and 98% were found at T4 and T8, respectively. Simultaneously,

TABLE IV. PLASMA LIPID CONCENTRATION DATA FROM SUBJECTS AT DIFFERENT TIME POINTS OF THE STUDY* Parameter TG (mM/L) TC (mM/L) LDL-C (mM/L) HDL-C (mM/L)

T⫺4

T0

T4

T8

2.35 ⫾ 0.24a 6.16 ⫾ 0.25a 4.23 ⫾ 0.25a 1.06 ⫾ 0.04

2.42 ⫾ 0.29a 6.03 ⫾ 0.23a 4.47 ⫾ 0.24a 1.07 ⫾ 0.05

1.96 ⫾ 0.14b 5.38 ⫾ 0.14b 3.51 ⫾ 0.14b 1.09 ⫾ 0.05

1.79 ⫾ 0.13b 5.60 ⫾ 0.15b 3.68 ⫾ 0.14b 1.18 ⫾ 0.06

* Average ⫾ standard error of the mean (n ⫽ 30). Values with different superscript letters are significantly different (P ⬍ 0.05). HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triacylglycerol; T⫺4, initial values; T0, after 4-wk consumption of semi-skimmed milk; T4, after 4-wk consumption of enriched milk; T8, after 8-wk consumption of enriched milk

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TABLE V. PLASMA VITAMIN E, TAC, MALONDIALDEHYDE, OXIDIZED LDL, AND LAG TIME MEASURED IN LDL PARTICLES ISOLATED FROM SUBJECTS AND CONCENTRATIONS OF PLASMA ICAM-1, VCAM-1, AND LP(A) MEASURED IN PLASMA* Parameter Vitamin E (␮M/L) TAC (␮M Trolox) Lag time (min) Malondialdehyde (␮M/L) Oxidized LDL (U/L) Vitamin E (␮M/L)/TG (mM/L) VCAM-1 (␮g/L) ICAM-1 (␮g/L) Lp(a) (mg/100 mL)

T⫺4

T0

T4

T8

99.82 ⫾ 6.40 173,63 ⫾ 2.75 86.32 ⫾ 3.01 0.767 ⫾ 0.126 28.47 ⫾ 2.82 22.01 ⫾ 2.02a 664 ⫾ 36a 212 ⫾ 13.5 36.64 ⫾ 5.57

95.27 ⫾ 6.09 174,53 ⫾ 2.70 85.08 ⫾ 2.72 0.966 ⫾ 0.093 24.60 ⫾ 2.45 18.43 ⫾ 2.01a 848 ⫾ 65b 203 ⫾ 14 32.32 ⫾ 4.86

105.50 ⫾ 6.98 173,88 ⫾ 2.69 89.76 ⫾ 2.49 0.929 ⫾ 0.084 ND 26.54 ⫾ 2.33b 488 ⫾ 47c 234 ⫾ 14 34.05 ⫾ 5.30

105.41 ⫾ 6.00 172,99 ⫾ 2.62 82.36 ⫾ 2.49 1.028 ⫾ 0.144 24.97 ⫾ 2.17 28.08 ⫾ 2.11b 585 ⫾ 33d 229 ⫾ 17 33.49 ⫾ 5.08

* Average ⫾ standard error of the mean (n ⫽ 30). Values with different superscript letters are significantly different (P ⬍ 0.05). ICAM-1, intercellular adhesion molecule-1; LDL, low-density lipoprotein; Lp(a), lipoprotein(a); ND, not determined; TAC, total antioxidant capacity; T⫺4, initial values; T0, after 4-wk consumption of semi-skimmed milk; T4, after 4-wk consumption of enriched milk; T8, after 8-wk consumption of enriched milk; VCAM-1, vascular cell adhesion molecule-1

plasma levels of tHcy decreased significantly by 16% and 18% (P ⬍ 0.05) at T4 and T8, respectively, compared with levels detected at T⫺4. The decrease was more prominent during the first 4-wk consumption of the enriched milk and was sustained for the next 4 wk. In this sense, the increase in plasma folate concentration was also more pronounced during the first 4 wk (Figure 1).

DISCUSSION The influence of enriched milk consumption on risk factors of CVD in middle-age hyperlipidemic subjects was studied. The 8-wk administration of the enriched milk resulted in significant 20% and 33% increases in plasma levels of DHA and EPA, respectively, demonstrating that compliance with the consumption of the product was good. Absorption of EPA and DHA from fish oil increases when associated with other fats and is spread out in small doses during the day.18 The fact that milk fat is highly dispersed in very small micelles, thereby increasing the surface of absorption of fats and lipid-soluble compounds,19 may explain the significant increases in plasma levels of DHA and EPA when only small amounts were supplemented in the diet through the enriched milk.

FIG. 1. Plasma folate (squares) and plasma total homocysteine concentrations (triangles) at different time points in the study (n ⫽ 30). *Significantly different from T⫺4 (P ⬍ 0.05). Hcy, fasting homocysteine; T⫺4, initial values; T0, after 4-wk consumption of semi-skimmed milk; T4, after 4-wk consumption of enriched milk; T8, after 8-wk consumption of enriched milk.

The prevalence of hypercholesterolemia in Spain (defined as total cholesterol ⬎ 5.2 mM/L) in the age range 35 to 64 y is about 60% (56.7% in men and 58.6% in women). In addition, approximately 20% of the Spanish population have cholesterol levels above 6.5 mM/L.20 We believe that changes in lifestyle and dietary patterns, with the very extended use of convenience foods, are responsible for the prevalence of hypercholesterolemia in Spain. Plasma concentrations of total cholesterol, LDL cholesterol, HDL cholesterol, and triacylglycerols did not differ significantly between men and women at the beginning of the study (Table IV). We advertised for subjects within the age range of 45 to 65 y, preferably with high levels of blood triacylglycerols, who were likely to have blood lipid values in the moderate to high range. The initial concentrations at baseline were beyond reference values reported for normolipidemic subjects (total cholesterol ⬎ 6.21 mM/L; LDL cholesterol ⬎ 4.14 mM/L; triacylglycerols ⬎ 2.25 mM/L).17 The 8-wk consumption of the enriched milk decreased plasma triacylglycerol levels to normal (⬍1.70 mM/L). In addition, total and LDL cholesterol concentrations at the end of the intervention were close to normal (total cholesterol ⬍5.17 mM/L; LDL cholesterol ⬍ 3.36 mM/L). The lipid-lowering effect was more prominent during the first 4-wk consumption of the enriched milk and maintained for the next 4 wk. We previously reported the cardiovascular effects derived from consumption of this enriched milk in young normolipidemic subjects.21 In that study, consumption of the milk decreased plasma cholesterol concentration but did not affect triacylglycerol values. In contrast, a similar study in normolipidemic subjects reported decreases of up to 19% in plasma triacylglycerol values and a 19% increase in HDL cholesterol levels after 6-wk consumption of milk enriched with similar amounts of ␻-3 PUFAs.19 Similar effects, i.e., decreased triacylglycerols and increased HDL in plasma, have been reported by other investigators22 over a 5-wk period of daily administration of pharmacologic amounts of PUFAs (4.5 g DHA plus EPA) in capsules. These results showed that the vehicle of administration also plays a role in the effects produced. The lowering effects of oleic acid on total and LDL cholesterol levels have been described extensively and are associated with a wide range of physiologic effects. However, this study was carried out in the context of a Mediterranean lifestyle in which the average intake of oleic acid is 23 g/d (Table I). In other populations and dietary patterns, administration of this enriched milk may produce a greater influence at this level.

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We addressed the question of whether regular intake of enriched milk used in this study, because of its PUFAs content, would make plasma and LDL particles more prone to oxidation or whether it would decrease levels of endogenous antioxidants with respect to possible negative effects. In our study, consumption of the enriched milk did not increase any of the LDL or plasma oxidation parameters analyzed (Table V). Possible explanations for these results may be due to the amount of PUFAs being too small to induce changes in plasma oxidation or to a compensatory effect derived from vitamin E supplementation, which may have counteracted the oxidation effects. Schnell et al.23 suggested that vitamin E may reduce lipoprotein oxidation susceptibility in vivo when administered with PUFAs. Although vitamin E was supplemented in the enriched milk to provide approximately 75% of the recommended daily allowance, we detected no significant increase in plasma vitamin E concentration. Other research studies have reported that much higher levels of vitamin E, supplemented in milk or in capsules, are needed to produce a significant increase in plasma levels.24,25 One possible explanation may be the remarkable decrease in blood lipids found in our study that also may have limited the concentration of vitamin E in plasma. The actual ratio of vitamin E to plasma lipids increased significantly (Table V). The ratio of vitamin E to plasma lipids (triacylglycerol or cholesterol) recently has been proposed as a better oxidative stress marker than plasma vitamin E alone.26 Our results are in accordance with other investigators reporting that ␻-3 PUFAs in moderate amounts do not increase LDL oxidation when provided in the context of a diet rich in monounsaturated fatty acids.27 Hyperhomocysteinemia is an important risk factor of atherosclerosis. Possible mechanisms for homocysteine-induced atherosclerosis include endothelial dysfunction, promotion of lipoprotein oxidation, and increased cholesterol synthesis in hepatocytes.28 Homocysteine-lowering effects of folic acid and vitamin B6 have been well documented. Although folic acid has been described as the main nutrient responsible for decreased tHcy, the addition of vitamins B12 and B6 to folic acid supplements or enriched foods may maximize the decrease of homocysteine.29 Intake of 500 mL/d of the enriched milk contributed more than 70% of the Expert Group on Vitamins and Minerals recommended daily allowances of folic acid and vitamins B12 and B6.30 Subjects varied from a suboptimal folate status (i.e., plasma folate ⬍15 nM/L)31 at the beginning of the study to an optimal folate status (18 nM/L at T8) after supplementation with enriched milk. Average tHcy plasma concentration of subjects (17.72 ␮M/L) indicated slight hyperhomocysteinemia (normal range, 5–15 ␮M/L).32 The 8-wk supplementation with the enriched milk produced a significant 18% decrease, which restored tHcy levels to within the normal range (14,69 ␮M/L) A major role for VCAM-1 but not for ICAM-1 has been found in early atherosclerosis.33 VCAM-1 and ICAM-1 are expressed by aortic endothelium in regions predisposed to atherosclerosis and are upregulated in hypercholesterolemic animals. However, their expression patterns are different, suggesting different functions for these molecules in lesion initiation and different mechanisms that are not equally sensitive to ␻-3 PUFA-34 or Hcy-related interventions.35 This may explain the different behaviors of VCAM-1 and ICAM-1 in our study. The significant decrease in VCAM-1 is in agreement with other studies linking ␻-3 fatty acids to decreases in soluble markers of endothelial function.21,35 The plasma concentration of ICAM-1 did not change throughout our study. A recent study reported that fish oil supplementation (1.2 g EPA plus DHA) in human subjects for 12 wk significantly decreased (20%) plasma VCAM-1 concentration, whereas ICAM-1 concentration was not affected.4 High concentration of blood Lp(a) has been proposed as an independent risk factor of CVD. Enriched milk consumption did not change Lp(a) concentration in our study in contrast to other ␻-3 PUFA intervention studies with similar doses.36

Nutrition Volume 20, Number 6, 2004 In this study we have shown that consumption of PUFAs, oleic acid, and vitamins administered in a dairy product may be an effective way to decrease risk factors of CVD. Supplemented food may play an important role in CVD prevention without involving major dietary changes in the population.34

ACKNOWLEDGMENTS The authors thank Jose A. Ferna´ ndez for help with blood extractions, Ruth Wilson for revising the English-language manuscript, and especially Carlos Rodriguez and Antonio D. Valero for technical assistance.

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